Citation to any reference in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this reference forms part of the common general knowledge or of the prior art in any country. All references cited herein are specifically incorporated herein by reference in their entirety.
The immune system enables a host organism to discriminate self from non-self antigens, as well as to recognize and eradicate invasive pathogens. The adaptive immunity system relies on highly polymorphic molecules, such as class I and class II antigens of the major histocompatibility complex (MHC), T cell receptors, and B cell receptors, to present antigens to T cells and B cells, thus leading to the activation of immune system. The mechanism by which the innate immunity system can recognize these diverse antigens remained unsolved until the emergence of the concept of ‘pattern recognition receptors (PPRs)’ proposed by Janeway (Janeway, 1989, Cold Spring Harb Symp Quant Biol 54 Pt 1, 1-13). This hypothesis was later proved correct by the identification of pathogen-associated molecular patterns (PAMPs) which are recognized by TOLL-like receptors (Aderem and Ulevitch, 2000 Nature 406, 782-7; Akira and Takeda, 2004, Nat Rev Immunol 4, 499-511; Athman and Philpott, 2004, Curr Opin Microbiol 7, 25-32), lectin receptors (Cambi and Figdor, 2003, Curr Opin Cell Biol 15, 539-46), immunoglobulin-like (Ig-like) receptors (Daws et al., 2003, J Immunol 171, 594-9), and NOD proteins (Athman and Philpott, 2004, Curr Opin Microbiol 7, 25-32), and others (Liu et al., 2001, J Biol Chem 276, 34686-94; McDonald et al., 2005, J Biol Chem 280, 20177-80). In addition to the well characterized PAMPs recognized by TOLL-like receptors (Akira and Takeda, 2004, Nat Rev Immunol 4, 499-511), recent study indicates that the host immune system can recognize invasive pathogens through specific carbohydrate antigens. For example, mannose receptors can recognize the high mannose sugar moiety expressed on the surface of pathogens (Stahl and Ezekowitz, 1998, Curr Opin Immunol 10, 50-5), while the Dectin-1 receptor can bind specifically to β-glucan, the major backbone of polysaccharides on fungus walls (Brown and Gordon, 2001, Nature 413, 36-7; Herre et al., 2004, Mol Immunol 40, 869-76). These results suggest that the carbohydrate structures associated with pathogens are one of the targets recognized by the innate immunity receptors of immune cells.
The funguses species Ganoderma and Cordyceps are the most popular herbal drugs taken in China to medicinal purposes. Polysaccharides extracted from Ganoderma lucidum (also known as Ling zhi, Reishi) have been used in traditional Chinese medicine as anti-tumor agents and as immuno-modulating agents (Lien, 1990, Prog Drug Res 34, 395-420; Wang et al., 2002, Bioorg Med Chem 10, 1057-62; Shiao, 2003, Chem Rec 3, 172-80), while those extracted from Cordyceps sinensis (Cordyceps, Caterpillar fungus) have been shown to alter apoptotic homeostasis, and to improve respiratory, renal, and cardiovascular functions (Buenz et al., 2005, J Ethnopharmacol 96, 19-29; Zhu et al., 1998, J Altern Complement Med 4, 289-303; Zhu et al., 1998, J Altern Complement Med 4, 429-57), as well as to increase whole body sensitivity to insulin (Balon et al., 2002, J Altern Complement Med 8, 315-23). However, the polysaccharide composition of the extracts vary when they the polysaccharides are extracted from different sources, from different strains, and under different growing conditions.
Analytical methods relying on high-performance liquid chromatography (HPLC) and proton-nuclear magnetic resonance have been applied to investigate the components of polysaccharides isolated from Ganoderma lucidum and Cordyceps sinensis (He and Seleen, 2004, Int. J. Med. Mushrooms 6, 253). However, the HPLC chromatogram is based on the comparison with ganoderic acid A and C (two major triterpenes of Ganoderma lucidum) or adenosine. It is still difficult to know whether the extracts contain the active components of polysaccharides based on the mass spectrum.
Cellular receptors are identified that induce plasma leakage and other negative effects when infected with flaviviruses, such as dengue virus or Japanese encephamyelitis virus. Using fusion proteins disclosed herein, the receptors to which a pathogen, such as flavivirus, binds via glycan binding are determined. Once the receptors are determined, the effect of binding to a particular receptor may be determined, wherein targeting of the receptors causing a particular symptom may be targeted by agents that interrupt binding of the pathogen to the receptor. Accordingly, in the case of dengue virus and Japanese encephamyelitis virus, TNF-α is released when the pathogen binds to the Dengue Virus Lectin Receptor 1 (DLVR1/CLEC5A receptor. Interrupting the DLVR1/CLEC5A receptor with monoclonal antibodies reduced TNF-α secretion without affecting secretion of cytokines responsible for viral clearance thereby increasing survival rates in infected mice from nil to around 50%.